The Laboratory Core will provide tiie centralized molecular microbiology diagnostic support, bacterial and cell culture support. Cytokine measurements, and training needed for each of the Projects and other Cores. Neisseria gononrhoeae (GC), Chlamydia trachomatis (Ct). Trichomonas vaginalis (TV), will be cultijre for diagnostic PCRs DNA positive controls and DNA sequencing controls. GC will be culture from animal secretions and tissue during in-vivo experiments to measure organism numbers (loads) in gene deficient (KO) mice compared to wild type mice. GC fluorescence staining will be use to evidence infection for in-vivo experiments, and tiie morphology of infected macrophages. The core will cultijre macrophages from wild type and knockout mice, to be challenged witii GC to study tiie role of nod- like receptors (NLRs). DNA will be extracted from mice vaginal secretions and tissue, collected at different time points during infection and GC DNA will be detected by PCR using specific primer. The organism loads contained in the sample will be quantified using a new end point quantitative PCR method (a-PCR) tiiat we have previously validated to study Ct infection and transmission using human genital specimens, and for the study of Francisella tularensis (Ft) infection in the mouse model using tissue samples (spleen, liver, lungs and kidneys). This assay has an excellent correlation with quantitative Ct or Ft culture. GC loads will be quantified in in-vivo and in-vitro experiments and compared to culture. The presence of a threshold in the number of organisms in secretions or tissue that divide mice into responder vs. non-responders to bacterial antigen stimulation will be investigated. We hypothesize that the innate response is dependent on the organism load, and that uninfected mice and mice with low organism loads will have a lower response (or no respond) compared with mice having higher organism loads. We will study how GC loads: 1) are modified by changes in innate immunity respond in infected KO mice and double KO mice); 2) how they conrelate with quantitative measurements of Cytokines, and 3) influence the development of sterility in infected mouse.